JP5638747B2 - Cell collection and isolation method and kit for genetic diagnosis of carcinoma - Google Patents

Description

  The present invention relates to a method and kit for collecting a target substance, and more specifically, to a method and kit for recovering and isolating cells that can be applied to pretreatment for genetic diagnosis of carcinoma. is there.

  In recent years, in the field of genetic diagnosis, particularly in the field of genetic diagnosis for definite diagnosis of cancer, attention has been focused on methods for recovering specific cells from test samples and cell populations (eg, blood, urine, stool) that contain a large amount of contaminants. Gathered.

  Cancer mortality has been the highest in Japan since 1981, accounting for 31.1% of all deaths in 2004. Carcinoma, which is a malignant tumor of epithelial cells, occupies most of the cancer and occurs in many tissues such as the stomach, lungs, large intestine, liver, and breast. Among them, the number of patients with colorectal cancer has increased rapidly in recent years and is expected to increase further in the future. On the other hand, unlike other cancers, colorectal cancer is known to be completely cured by surgery if detected early.

  Various tests have been devised for early detection of colorectal cancer. Currently, fecal occult blood and endoscopy are representative methods of colorectal cancer screening. The fecal occult blood test is performed as a general screening, and if it is positive, an endoscopy is performed for a definitive diagnosis. In the fecal occult blood test, the presence of hemoglobin in the stool is checked to confirm the presence of bleeding in the intestine and indirectly predict the occurrence of colorectal cancer. It contains a lot of false negatives. Endoscopy is highly sensitive and specific to the development of colorectal cancer because the inside of the large intestine is examined directly with an endoscope. However, it is not suitable for colorectal cancer testing for asymptomatic general people because it is highly invasive and may cause bleeding and perforation, and it may be painful.

  Therefore, in order to solve the above-mentioned problems, attention has recently been focused on a method for collecting cancer cells from feces, extracting DNA from the cells, and performing genetic testing as a general-purpose colorectal cancer screening method. In JP-A-2005-46062 (hereinafter referred to as the first conventional example), as a method for recovering cells from feces, a “solid carrier” in which “antibodies against surface antigens of epithelial cells and / or epithelial cancer cells” are immobilized. It has been attempted to collect “epithelial cells and / or epithelial cancer cells” from feces.

  As a method of dissociating cells collected with antibody-bound beads into antibody-bound beads and cells after cell recovery, Japanese Patent Application Laid-Open No. 9-508534 describes, “Antibody-mediated beads or other beads from a heterogeneous cell suspension. A “non-enzymatic method” is provided for releasing positively selected cells by binding to a solid support (hereinafter referred to as second conventional example). According to this, “the antibody that binds to the surface antigen present on the target cell” can be released from the cell surface antigen by binding to the antibody after the step of separating the target cell from the cell suspension. The antibody is dissociated from the recovered cells by adding a peptide, that is, a peptide containing the epitope sequence of the antibody. In this second conventional example, CD34 positive stem cells are selectively recovered from a bone marrow or blood suspension derived from a cancer patient, and the recovered “CD34 positive cell composition is used to reconstitute a patient's immune system. For use ".

  The first conventional example has the following problems. In the cells after collection, “an antibody against the surface antigen of epithelial cells and / or epithelial cancer cells”, that is, an anti-EpCAM antibody (clone name: BerEP4) remains bound. EpCAM molecules are cell surface antigens involved in cell adhesion and signal transduction, and it is known that the proportion of living cells decreases if this antibody remains bound on the cell surface (FIG. 5). In order to recover cells from a small amount of sample and efficiently recover nucleic acids used for genetic diagnosis from the cells, it is desirable to maintain the activity of the cells after recovery. However, in the first conventional example, since the antibody remains on the cell surface, the percentage of living cells is reduced and the nucleic acid is degraded, so that the yield of nucleic acid extraction is reduced and the nucleic acid that can be used as a template for PCR. There is a problem that it is difficult to secure a sufficient amount of the protein and high-precision genetic testing cannot be performed.

  In the second conventional example, after collecting cells with antibody-bound magnetic beads, the antibody-bound magnetic beads and cells are dissociated by adding a peptide containing the antibody epitope sequence. When combined with the first conventional example and applied to genetic diagnosis of carcinoma, the following problems are raised. In the second conventional example, only a recovery example of CD34 positive cells is shown, and the effect on other antigens is unknown. For example, if cancer cells are collected using the antibody against EpCAM antigen shown in the first conventional example, and the peptide containing the antibody epitope sequence is added to dissociate the cell from the antibody-bound magnetic beads, it can be purchased. Since there is no antibody whose epitope sequence is disclosed among anti-EpCAM antibodies, the peptide to be added is unknown and cannot be applied as it is. In addition, EpCAM antigen is known to increase in expression with canceration, but when high-expressing antigen is targeted for cell recovery, the antigen and antibody present on the magnetic bead surface Since interactions are thought to exist at a high frequency, antibodies and cells cannot be dissociated unless all of these interactions are eliminated by the addition of peptides.

JP2005-46062 Special table flat 9-508534

  The present invention provides a method for forming a conjugate with a target substance using a substance that binds to the collection target substance and recovering this, and then dissociating the collection target substance from the conjugate. It is an object to provide a method for efficiently dissociating bonds.

  In the step of dissociating the substance to be collected from the conjugate, the present inventors use a carrier that holds on its surface a probe that includes at least a part of a region that specifically binds to the substance that binds to the substance to be collected. The present inventors have found that the above problems can be solved and have completed the present invention.

That is, the present invention includes the following inventions.
(1) A method for collecting substances,
A first step of generating a conjugate by binding the first carrier having the first substance immobilized on the surface of the first support and the second substance, which is the substance to be collected;
A second step of collecting the conjugate based on the first support;
After the first step, a second carrier that holds on its surface a probe that includes at least part of a region that specifically binds to the first substance is supplied, and the first carrier and the second carrier The method further comprising: a third step of forming a complex of: and dissociating the second substance from the conjugate; and a fourth step of removing the complex to obtain the second substance.
(2) The collection method according to (1), wherein the first substance is an antibody.
(3) The collection method according to (2), wherein the second substance is a collection target substance containing an antigen recognized by the antibody.
(4) The collection method according to (3), wherein the second substance is a cell.
(5) The collection method according to (2), wherein the probe is a peptide including at least part of a region that specifically binds to the antibody.
(6) The collection method according to (5), wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1.
(7) A kit for collecting substances,
A first substance that specifically binds to the substance to be collected;
A first support for fixing the first substance to the surface;
A peptide comprising at least a part of a region that specifically binds to the first substance;
The kit having a second support for holding the peptide on the surface.
(8) The kit according to (7), wherein the first substance is an antibody.
(9) The kit according to (7), wherein the second support is a bead.
(10) The kit according to (8), wherein the peptide contains the epitope sequence of the antibody.
(11) The kit according to (10), wherein the antibody is an anti-EpCAM antibody, and the peptide includes an epitope sequence of the anti-EpCAM antibody.
(12) The kit according to (11), wherein the epitope sequence is the amino acid sequence of SEQ ID NO: 1.
(13) The kit according to (7), wherein the collection target substance is a cell.
(14) The kit according to (13), wherein the cell is a human epithelial cell.

  According to the present invention, in a method for forming a conjugate with a target substance by using a substance that specifically binds to the target substance to be collected and recovering the same, and then dissociating the target substance from the conjugate, It is possible to efficiently dissociate the strong bond in.

The components of the present invention will be described with reference to FIGS.
FIG. 1 shows a second substance 1 to be treated by the present invention, that is, a substance to be collected. Examples of substances to be collected include biomolecules such as cells, antibodies, antigens, proteins, peptides, sugars, and nucleic acids (including DNA and RNA). In the present invention, cells are preferably collected substances, that is, targets. Examples of cells include any cells (eg, spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow cells) of mammals (eg, humans, guinea pigs, rats, mice, rabbits, pigs, sheep, cows, monkeys, etc.). Mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils) Spheres, basophils, eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursors of these cells Cells) and blood cells (eg, white blood cells, red blood cells). The present invention is advantageous in the collection of cancer cells. For example, cancer cells of the above cells, as well as brain, stomach, pancreas, kidney, liver, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, large intestine) , Small intestine), prostate, testis, ovary and other tissues suitable for collecting cancer cells derived from cancer. A cell population obtained by once culturing cells derived from these tissues may be used. More specifically, cells expressing EpCAM molecules on the cell surface, such as HT29 cells, can be mentioned. The amino acid sequence of the EpCAM molecule is known and is registered as NP_002345 (SEQ ID NO: 2) in a public database (GenBank).

  FIG. 2 shows a first carrier 4 which is a component of the present invention. The first carrier 4 is composed of a first substance 2 and a first support 3. The first substance is not particularly limited as long as it is a substance that specifically binds to the second substance to be collected, and can be appropriately selected by those skilled in the art. Examples of the second substance include biomolecules such as antibodies, antigens, epitopes, cells, proteins, peptides, sugars, and nucleic acids (including DNA and RNA). In the present invention, an antibody is preferably used as the first substance. When a cell is targeted as a collection target substance, an antibody that specifically binds to an antigen expressed on the surface of the target cell is preferably used as the first substance. When targeting cancer cells, it is preferable to use an antibody that binds to an antigen specifically expressed on the surface of the cancer cells as the first substance. When collecting cells expressing EpCAM molecules on the cell surface, it is preferable to use the monoclonal antibody hrk29 produced by the hybridoma hrk29. Hybridoma hrk29 (Hybridoma hrk29) has been deposited under the accession number FERM P-21604 at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (1st, 1st, 1st East, 1st Street, Tsukuba, Ibaraki Prefecture).

  The first support 3 is not particularly limited as long as it can fix the first substance 2, and for example, a resin, a glass material, iron, iron oxide, or the like can be used. These may have any shape, and can be appropriately selected depending on the type of material and the subsequent steps. Examples thereof include a plate shape, a bead shape, a thin film shape, a thread shape, and a coil shape. If the beads are made of iron or iron oxide, magnetic separation is possible, and the subsequent recovery process can be carried out easily. The size of the first support 3 may be any size, and is determined by the type of material, the subsequent steps, etc., as with the shape. For example, if it is a magnetic bead, if it is about 1.0-4.5 micrometers, it will be suitable for operation of subsequent magnetic separation.

  As a method for binding the first substance 2 to the first support 3, when the antibody is bound to the first support 3, for example, a secondary antibody method can be used. Other coupling methods can also be employed. For example, as a method of directly binding to the first support 3, the first support 3 is activated by cyanogen bromide treatment or the like, and the first substance 2 having an amino group or a hydroxyl group (for example, an antibody) ). As a method of indirectly binding, for example, a method of binding the first substance 2 (for example, an antibody) through the first support 3 with the protein A, protein G or the like added on the surface thereof is also available. Can be used. By using the first support 3 having streptavidin added to the surface, the first substance 2 can be biotinylated to similarly bind indirectly.

The amount of the first support 3 used in the production of the first carrier is preferably in the range of 1 to 6 × 10 ⁶ . When an antibody is used as the first substance 2, the amount of the antibody can be used in the range of 0.03 to 0.8 μg, but 0.3 μg or more is desirable. A fixed amount of the first support 3 is washed once or a plurality of times with a buffer solution such as PBS, and then suspended in a certain amount of PBS and the antibody is added. The reaction time between the antibody and the magnetic beads is preferably about 30 minutes to 1 hour. After reacting for a certain period of time, the first carrier 4 in which the antibody is immobilized on the first support 3 can be obtained by washing once or a plurality of times with a buffer solution such as PBS.

  FIG. 3 is a schematic diagram of the second carrier 7. The second carrier 7 includes a probe 5 including at least part of a region that specifically binds to the first substance, and a second support 6.

  The second support 6 is not particularly limited as long as the probe 5 can be fixed. For example, a resin, a glass material, iron, iron oxide, or the like can be used. These may have any shape, and can be appropriately selected depending on the type of material and the subsequent steps. Examples thereof include a plate shape, a bead shape, a thin film shape, a thread shape, and a coil shape. If the beads are made of iron or iron oxide, magnetic separation is possible, and the subsequent recovery process can be carried out easily. The size of the second support 6 may be any size, and is determined by the type of material, the subsequent steps, etc., as with the shape. For example, in the case of magnetic beads, the thickness is preferably about 1.0 to 4.5 μm, more preferably about 1.0 μm.

  When an antibody is used as the first substance, a peptide containing an epitope sequence recognized by the antibody is preferably used as the probe 5. When the monoclonal antibody hrk29 produced by the hybridoma hrk29 is used as the antibody, a peptide containing the amino acid sequence of SEQ ID NO: 1 (ICSKLA) is preferably used as the epitope sequence. The amino acid sequence of SEQ ID NO: 1 corresponds to the 58th to 63rd amino acids of the amino acid sequence of the EpCAM molecule (SEQ ID NO: 2). The number of residues of the peptide is not particularly limited, but is preferably 11 residues or more and 15 residues or less. More preferably, it is a partial sequence of the amino acid sequence of SEQ ID NO: 2, and a peptide consisting of a sequence of 11 to 15 residues including the 58th to 63rd amino acids of SEQ ID NO: 2 is used as the probe 5.

  As the peptide, a chemically synthesized peptide or a gene engineered peptide can be used. If it has a biotin label at either the N-terminus or the C-terminus, the subsequent binding to the second support 6 becomes easy. As a method of binding the peptide to the second support 6, for example, in the second support 6 with streptavidin added to the surface, it is possible to bind the peptide indirectly by biotinylating the peptide. is there. The amount of peptide used is preferably in the range of 40 to 400 pmol. A certain amount of the second support 6 is washed once or a plurality of times with a buffer solution such as PBS, and then suspended in a certain amount of PBS and the peptide is added. The reaction time between the peptide and the second support 6 is preferably about 30 minutes to 1 hour. After reacting for a certain period of time, the second carrier 7 can be obtained by washing once or a plurality of times with a buffer solution such as PBS.

  The outline of the method of the present invention will be described with reference to FIG. FIG. 4 is a flowchart showing operations in the method of the present invention. First, the components of the present invention will be described with reference to FIG. The conjugate 8 is a combination of the second substance 1 (for example, a target cell) and the first carrier 4, and the magnet 9 is used for magnetic separation. The purified conjugate 10 is a conjugate 8 obtained after removing impurities, and the complex 11 is a composite of the first carrier 4 and the second carrier 7.

  The method of the present invention includes four steps of Step 1, Step 2, Step 3, and Step 4. Step 1 is a step of forming a conjugate, specifically, a step of mixing the second substance 1 (for example, a target cell) and the first carrier 4 to form a conjugate 8. Step 2 is a step of collecting the conjugate. Specifically, the conjugate 8 is magnetically separated using the magnet 9 to remove impurities other than the conjugate 8 to obtain a purified conjugate 10. Step 3 is a step of complex formation and conjugate dissociation. Specifically, the purified conjugate 10 and the second carrier 7 are mixed to form a complex 11, thereby collecting from the purified conjugate 10. This is a step of dissociating the second substance 1 (for example, the target cell) that is the target substance. Step 4 is a step of removing the complex. Specifically, the complex 11 is removed by magnetic separation, and the second substance 1 (for example, target cell) is recovered.

  An embodiment in which cells are collected as the second substance 1, an antibody is used as the first substance 2, and a peptide containing an epitope sequence recognized by the antibody is used as the probe 5 will be described below.

The number of cells used in Step 1 is not particularly limited, but can be suitably used in the range of 1 × 10 ⁴ to 1 × 10 ⁶ cells. The reaction time between the target cell 1 and the first carrier 4 in step 1 is not particularly limited, but is preferably about 30 minutes to 1 hour. In step 2, the first carrier 4 brought into contact with the target cell 1 is collected using a magnet 9 and washed once or a plurality of times using a buffer solution that does not adversely affect the cells such as PBS. Thereafter, in step 3, the second carrier 7 is added, and the first carrier 4 is dissociated from the target cells 1 by stirring for a certain time. The reaction time is not particularly limited, but is preferably about 30 minutes to 1 hour. The ratio of the first carrier 4 and the second carrier 7 is not particularly limited, but the amount of the second carrier can be added so that the peptide 5 having the number of moles or more of the antibody 2 present on the surface of the first support 3 can be added. Preferably 7 is added. After the first carrier 4 and the target cell 1 are dissociated, the target cell 1 dissociated from the first carrier 4 can be obtained by removing the complex 11 using the magnet 9 as step 4.

  Note that the main aspect of the present invention is to recover the collection target substance 1 from a sample containing a large amount of contaminants and dissociate the first carrier 4 used for the collection from the collection target substance 1 after the recovery. Although subsequent operations are not described in detail, for example, by applying the method described in Conventional Example 1, cancer genetic diagnosis can be performed through nucleic acid extraction, PCR, gene mutation analysis, and the like.

  According to the present invention, when an antigen highly expressed on the cell surface is used as a target for cell recovery, a strong bond between the antibody and the cell can be efficiently dissociated. In the present invention, unlike the first conventional example, since no antibody is present on the cell surface after cell recovery and isolation, cell activity can be maintained. Further, unlike the second conventional example, the use of a carrier that retains a peptide containing an epitope sequence on the surface makes it possible to efficiently dissociate antibodies from cells. These make it possible to collect highly active cells with high purity, and to extract a sufficient amount of high-quality nucleic acid from the collected cells, which is highly accurate in genetic testing for carcinomas. Can be provided.

  In addition, by using a newly developed antibody with the epitope sequence clarified, it becomes possible to dissociate the anti-EpCAM antibody from the EpCAM molecule as an antigen using a peptide containing the epitope sequence of the antibody. Furthermore, by using a carrier that holds the peptide containing the epitope sequence on the surface, it becomes possible to efficiently dissociate the antibody and the magnetic beads bound thereto from epithelial cells that highly express EpCAM.

Hereinafter, specific examples of the present invention will be described. However, the present invention is not limited to these examples.
Example 1
Preparation of Antibody 2 Hybridoma was prepared according to a conventional method. EpCAM molecules prepared by gene recombination were mixed with adjuvant (TiterMax), and then Balb / C mice were immunized by the footpad method. After three immunizations, the lymph nodes were removed and lymphocytes and myeloma cells (P3U1) were fused to prepare a hybridoma. Hybridoma screening was performed using the amount of binding to the HT29 cell surface as an index. 100 μL of hybridoma culture supernatant was added to 1 × 10 ⁶ HT29 cells and allowed to stand at 4 ° C. for 30 minutes. After washing with PBS, 2 μg of goat-derived anti-mouse Ig antibody-PE label was added and again allowed to stand at 4 ° C. for 30 minutes. After washing with PBS again, it was suspended in 500 μL of PBS, and the fluorescence intensity was measured using a flow cytometer. Hybridomas that were positive in the screening were cloned twice by limiting dilution. After cloning, screening was performed again with a flow cytometer, and positive clones were selected. The clone name of the hybridoma thus obtained and the antibody produced by it was designated as hrk29. Hybridoma hrk29 (Hybridoma hrk29) has been deposited at the Patent Biological Depositary Center of the National Institute of Advanced Industrial Science and Technology (Higashi 1-chome, 1-chome, 1-chome, Tsukuba-shi, Ibaraki) as deposit number FERM P-21604. In order to know the immunoglobulin subclass of the monoclonal antibody hrk29, the subclass was determined using Mouse Monoclonal Antibody Isotyping Test Kit (Serotec). As a result, the isotype of monoclonal antibody hrk29 was found to be IgG1.

Epitope sequencing of antibody 2 To analyze the epitope sequence recognized by the monoclonal antibody hrk29, fragmented EpCAM molecules (EpCAM amino acid residues 24-62, 63-139, 24-139 / EC1, EC2, EC3, Figure 6) was expressed in E. coli, and the domain recognized by the monoclonal antibody hrk29 was examined by Western blot. As shown in FIG. 9, reactivity was observed for EC1 (molecular weight approximately 35 kDa) and EC3 (43 kDa). That is, it was confirmed that this antibody recognizes a region containing amino acids 24 to 62 of EpCAM.

  According to the amino acid sequence of EC1, peptides shown in FIGS. 10 and 11 (peptides A to E; SEQ ID NOs: 3 to 7, respectively) were synthesized, and the epitope sequence recognized by the monoclonal antibody hrk29 was determined to be ICSKLA (SEQ ID NO: 1) by ELISA. (Figure 12).

Production of the first carrier 4 The first carrier 4 was produced as follows. As the first support 3, Dynabeads (registered trademark) M-450 Sheep anti-mouse IgG was used, and as the antibody 2, the anti-EpCAM antibody prepared above: hrk29 was used. The first support 3 is washed once with PBS, suspended in 100 μL of PBS, 0.03 μg of antibody 2 is added to 1 × 10 ⁶ first supports 3, and stirred at room temperature for 30 minutes. did. Thereafter, the plate was washed twice with PBS and then suspended in 100 μL of PBS to obtain a first carrier 4.

Production of second carrier 7 A production method of the second carrier 7 having the peptide 5 on the surface is described below. Biotinylated peptide (amino acid sequence ICSKLAANKMKAE; SEQ ID NO: 8) containing Dynabeads (registered trademark) MyOne Streptavidin T1 as the second support 6 and the epitope sequence ICSKLA recognized by the antibody 2 (hrk29) prepared in this example as the peptide 5 ) Was used. The second support 6 is washed once with PBS, then suspended in 100 μL of PBS, 400 pmol of peptide 5 is added to 7 × 10 ⁸ beads, and stirred at room temperature for 30 minutes to contain the epitope sequence. A second carrier 7 holding the peptide 5 on the surface was obtained.

Cell recovery and dissociation Cell recovery and dissociation methods using magnetic beads with anti-EpCAM antibody bound to the surface are described below. 1 × 10 ⁶ HT29 cells, which are target cells 1, were dispersed in 500 μL of PBS. As Step 1, the first support 4 was added to this starting material and stirred at room temperature for 30 minutes. As Step 2, after the magnetic separation, the purified complex 10 was obtained after washing twice with PBS containing 0.5% BSA and 2 mM EDTA. In Step 3, the second carrier 7 was added and stirred at room temperature for 30 minutes. Thereafter, magnetic separation was performed as step 4 to collect the target cells 1 and to remove the complex 11 and the unreacted purified conjugate 10. From the number of collected target cells 1 and the number of target cells 1 contained in the purified conjugate 10, the ratio of target cells 1 dissociated from antibody 2 was calculated.

(Example 2)
Although it was the same method as Example 1, Qdot (registered trademark) 565ITKTM Streptavidin Conjugate was used as the second support 6 used for the second carrier 7.

  The results of Example 1 and Example 2 will be described with reference to FIG. FIG. 13 is a graph showing the ratio of the dissociated target cells 1, that is, the cell dissociation rate. In addition, the method in which the second conventional example was modified, that is, the result of adding only the peptide 5 instead of the second carrier 7 in Step 3, was also compared. As a result of using the second carrier 7, the ratio of the dissociated target cells 1 is increased as compared with the modified example of the second conventional example, that is, the method of adding only the peptide 5, and the effectiveness of the present invention is shown. It was. In other words, unlike the method in which only the peptide is added, by adding the second carrier 7, the target cell 1 and the antibody 2 can be dissociated more efficiently, and highly active cells are recovered with high purity. It became possible. This makes it possible to extract a sufficient amount of high-quality nucleic acid from the collected cells, and to provide high accuracy in genetic testing for carcinomas.

It is a figure which shows the 2nd substance (target cell) 1 among the components of this invention. FIG. 3 is a diagram showing a first carrier 4 among the components of the present invention. FIG. 3 is a diagram showing a second carrier 7 among the components of the present invention. It is a schematic diagram which shows one Embodiment of the method of this invention. It is a graph which shows the influence which an antibody has on a viable cell rate. FIG. 4 is a schematic diagram showing EC1, EC2, and EC3 prepared to determine the antigen domain of monoclonal antibody hrk29. It is a figure which shows the vector used for the expression of EC1, EC2, and EC3. It is a schematic diagram which shows the recombinant protein expressed with the vector of FIG. It is a figure which shows the domain of the antigen which monoclonal hrk29 recognizes. FIG. 2 shows peptides used to determine the epitope sequence. It is a figure which shows the arrangement | sequence of the peptide AE used in the Example. It is a figure which shows the reactivity of the monoclonal antibody hrk29 and peptides A-E. It is the figure which compared the cell dissociation rate at the time of using the modification of the conventional Example, and the method of this invention.

Explanation of symbols

1… Second substance (target cell)
2… First substance (antibody)
3 ... first support
4 ... first carrier
5 ... Probe (peptide)
6 ... second support
7 ... second carrier
8 ... Conjugate
9 ... Magnet
10 ... Purified conjugate
11 ... complex
12… Glutathione S-transferase
13… 6 × His tag
14 ... EC1 ~ 3

Claims (6)

A method of collecting cells expressing EpCAM molecules on the cell surface ,
The first carrier that binds the first carrier having the anti-EpCAM antibody immobilized on the surface of the first support and the cells expressing the EpCAM molecule on the cell surface, which is the collection target substance. Steps,
A second step of collecting the conjugate based on the first support;
After the first step, a second carrier holding a peptide containing the epitope sequence of the anti-EpCAM antibody on the surface is supplied to form a complex of the first carrier and the second carrier, and the binding The method comprising: a third step of dissociating the cell from the body; and a fourth step of removing the complex and obtaining the cell. The collection method according to claim 1 , wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1. A kit for collecting cells expressing EpCAM molecules on the cell surface ,
An anti-EpCAM antibody,
A first support for immobilizing the anti-EpCAM antibody on a surface;
A peptide comprising the epitope sequence of the anti-EpCAM antibody;
The kit having a second support for holding the peptide on the surface. The kit according to claim 3 , wherein the second support is a bead. The kit according to claim 3 or 4 , wherein the epitope sequence is the amino acid sequence of SEQ ID NO: 1. The kit according to any one of claims 3 to 5 , wherein the cells are human epithelial cells.

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